Innovating Urban Mosquito Control: Introducing Human-Controlled Breeding Sites as a Component of Integrated Mosquito Management (IMM)

6 This study introduces a novel method for mosquito control tailored specifically for urban and 7 suburban areas, addressing their unique challenges. The concept of Human-Controlled Breeding 8 Sites (HCBS) is presented as an innovative approach to reducing mosquito populations by 9 providing controlled oviposition sites within households. The paper is structured into three main 10 components. The first details the meticulous development of the HCBS method, the second 11 explores the intricate development of the HCBS device, crucial for effective implementation of 12 the HCBS approach. Finally, the experiment validating the HCBS methodology is described, 13 offering insights into its practical application and efficacy in mosquito population control. Our 14 proposal emphasizes the importance of considering mosquito needs, behavior and preferences in 15 designing effective mosquito control strategies. Gravid mosquitoes must find a suitable place for 16 oviposition, and HCBS provides an easy-access controlled environment for them to do so within 17 households. Contrary to the commonly current recommendation to destroy all possible locations 18 where mosquitoes can lay their eggs, the HCBS methodology provides an opportunity for gravid 19 mosquitoes to oviposit their eggs without being deterred by chemical odors or dead larvae or 20 pupae. It involves waiting for a timespan before effectively collecting and destroying the 21 individuals in one concerted effort with zero impact on the environment and at minimal cost. 22 Successful deposition of eggs by gravid mosquitoes into HCBS devices, followed by their 23 destruction within the designated timeframe, validates the system's efficacy in disrupting the 24 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 2 mosquito life cycle. Moreover, our findings demonstrate the significant influence of color 1 selection on HCBS effectiveness, with green HCBS devices attracting the highest number of 2 gravid female mosquitoes for oviposition. These integrated components offer a comprehensive 3 understanding of the HCBS approach, effectively bridging the gap between methodological 4 development, device design, and practical application. The findings underscore HCBS as a 5 valuable addition to mosquito control strategies, with potential applications in diverse 6 environments. Further research is essential to delve into and model the long-term effectiveness of 7 HCBS, aiming to optimize its design for maximum efficacy and scalability. This includes 8 studying its performance over extended periods and refining the design parameters to enhance its 9 functionality and widespread applicability including the use of disaster risk management 10 systems. 11

Mosquito control, Human-Controlled Breeding Sites (HCBS), Integrated Mosquito 12 Management, Oviposition sites, Vector control methods, Gravid female mosquitoes, Mosquito life 13 cycle, Public health 14 1. INTRODUCTION: 15 Climate change has emerged as a significant driver of mosquito population dynamics, 16 contributing to the expansion of mosquito habitats into new geographic regions (Mora, et al., 17 2022). Rising temperatures, altered precipitation patterns, and changes in humidity create 18 favorable conditions for mosquito breeding and survival, facilitating their spread into previously 19 unaffected areas (Lafferty, 2009). As a result, regions that were once considered free from 20 mosquito-borne diseases are now at risk of transmission due to the establishment of mosquito 21 populations in these newly suitable habitats. 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 3 Urbanization exacerbates the impact of climate change on mosquito-borne disease 1 transmission by creating environments conducive to mosquito proliferation. Rapid urban growth, 2 particularly in tropical and subtropical regions, leads to the development of densely populated 3 urban and suburban areas with abundant breeding sites for mosquitoes, such as discarded 4 containers, sewage systems, and stormwater drains (Ananya and Miller, 2021; Guagliardo et al., 5 2015). The convergence of increased human population density and expanding mosquito 6 populations in urban settings creates ideal conditions for the transmission of diseases like 7 Dengue fever, Malaria, West Nile virus and Zika virus. 8 Traditional mosquito control methods, primarily developed for rural environments with 9 low population density, face significant challenges in urban and suburban areas. The widespread 10 use of chemical insecticides may be impractical or environmentally undesirable in densely 11 populated urban settings, while biological control methods may be less effective due to the 12 limited availability of natural habitats for mosquito predators and parasites (Chadee et al., 2009). 13 Moreover, the heterogeneous and dynamic nature of urban landscapes poses challenges for 14 implementing source reduction and habitat modification strategies. 15 Addressing the growing threat of mosquito-borne diseases in urban and suburban areas 16 requires the development of innovative and context-specific control methods tailored to urban 17 environments. Integrated approaches that combine novel vector control technologies with 18 community engagement and public health interventions offer promising solutions for mitigating 19 disease transmission in urban settings (Muller et al., 2020). These approaches may include the 20 use of novel larvicides targeting mosquito breeding sites in urban landscapes, deployment of 21 spatial repellents to protect high-risk populations, and implementation of community-based 22 surveillance and response systems to detect and control disease outbreaks. 23 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 4 Efforts to develop and implement new mosquito control methods for urban and suburban 1 conditions must consider the social, economic, and environmental factors that influence disease 2 transmission dynamics in these settings (Bazin and Williams, 2018). Multidisciplinary 3 collaborations between researchers, policymakers, public health authorities, and communities are 4 essential for designing effective and sustainable strategies to reduce the burden of mosquito-5 borne diseases in urban areas. 6 The objectives of the present study are: 7 1. To introduce a novel method for mosquito control specifically designed for urban and 8 suburban areas, addressing the unique challenges these environments present. 9 2. To present the concept of Human-Controlled Breeding Sites (HCBS) as a new, innovative 10 approach to mosquito population reduction by providing a controlled environment for 11 mosquito females to oviposit their eggs within households. 12 3. To describe the design and functionality of the Human-Controlled Breeding Site device, 13 emphasizing its potential as a supplementary component of Integrated Mosquito 14 Management (IMM) strategies. 15 4. To investigate the oviposition preferences of mosquito females by testing multiple colors 16 within the Human-Controlled Breeding Sites devices, aiming to identify the most 17 attractive color for egg-laying and optimize the effectiveness of the control method. 18 This paper is structured into three main components. In Section 3.1, we detail the 19 meticulous development of the new HCBS method, laying the foundational groundwork for 20 substantiating the subsequent exploration and validation of the HCBS methodology. Section 3.2 21 delves into the intricate development of the HCBS device, an essential component for 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 5 implementing the HCBS approach effectively. Finally, Section 3.3 is dedicated to describing the 1 experiment conducted to validate the HCBS methodology, offering insights into its practical 2 application and efficacy in mosquito population control. Together, these integrated components 3 provide a comprehensive understanding of the HCBS approach, effectively bridging the gap 4 between methodological development, device design, and practical application. 5 2. LITERATURE REVIEW: 6 Mosquito-borne diseases pose significant threats to public health, particularly in tropical 7 and subtropical regions worldwide. Among the most notorious mosquito-borne illnesses are 8 Dengue fever, Malaria, Yellow Fever, West Nile virus and Zika, which collectively cause 9 millions of cases and thousands of deaths each year (World Health Organization, 2020). 10 Controlling the populations of disease-carrying mosquitoes is crucial for reducing the 11 transmission and burden of these diseases. In this literature review, we explore the biology and 12 behavior of mosquitoes, existing control methods, factors influencing mosquito attraction, and 13 the relationship between mosquito species and disease transmission. 14 2.1 Mosquito Biology and Behavior 15 Mosquitoes, belonging to the Culicidae family, are small, flying insects characterized by 16 their slender bodies, long legs, and elongated mouthparts adapted for piercing and sucking blood 17 (Clements, 1999). The life cycle of mosquitoes consists of four stages: egg, larva, pupa, and 18 adult. 19 Male and female mosquitoes exhibit significant differences in their lifespans. Male 20 mosquitoes typically live for up to 10 days (Clements, 1999, Lambert et. al. 2022), while females 21 can survive for a few weeks (Foster and Walker, 2009). Some sources suggest that males live for 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 6 1-2 weeks (Service, 2012), and females typically live anywhere from two to four weeks (Scott 1 and Takken, 2012). 2 The primary reason for this difference in lifespan is the distinct roles each gender plays 3 within the species. Males primarily live to mate and do not play a major role in the reproductive 4 cycle after mating (Service, 2012). On the other hand, female mosquitoes require extended 5 periods of life to lay eggs (Clements, 1999). 6 Moreover, the feeding habits of male and female mosquitoes also contribute to their 7 lifespan differences. Male mosquitoes feed only on plant nectar and sugary liquids, such as fruit 8 juices and honeydew (Clements, 1999). In contrast, female mosquitoes feed on blood in addition 9 to plant nectar, which provides the necessary nutrients for egg production (Foster and Walker, 10 2009). 11 Therefore, the differences in lifespan between male and female mosquitoes can be 12 attributed to their distinct roles in reproduction and their different feeding habits (Clements, 13 1999; Foster and Walker, 2009; Service, 2012; Scott and Takken, 2012), although a new study 14 suggest the lifespan difference can be less (Lambert et al., 2022). 15 Environmental factors significantly influence mosquito biology and behavior, with 16 temperature, humidity, and the availability of breeding sites playing crucial roles in shaping 17 mosquito population dynamics and distribution (Hugo et al., 2014; Paaijmans et al., 2009). 18 Human-related factors, such as urbanization (Durrance-Bagale et al., 2024) and changes in land 19 use, can also have a substantial impact on mosquito habitats and abundance (Gubler, 2011; 20 Leisnham et al., 2014). Additionally, characteristics like water type (Sumba et al., 2008), larvae 21 density (Allan and Kline, 1998; Gimnig et al., 2002; Kiflawi et al., 2003; Koenraadt and Takken, 22 2003), and even pheromone emissions by conspecific larvae (Chadee, 1993; Sumba et al., 2008) 23 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 7 influence oviposition behavior. However, a common thread among these factors is the necessity 1 of water in all oviposition sites. 2 2.2 Mosquito Control Methods 3 The World Health Organization (WHO, 2009) advocates for integrated vector 4 management (IVM) and suggests four major strategies: Chemical control involves the use of 5 insecticides to kill mosquito larvae and adults. These variously comprise fogging, residual 6 spraying, larviciding, and autodissemination. However, the emergence of insecticide resistance 7 poses challenges to the effectiveness of chemical control efforts (Hemingway and Ranson, 8 2000). 9 Biological control methods harness natural enemies of mosquitoes, such as predators and 10 parasites, to reduce mosquito populations. Predatory fish, such as Gambusia affinis (mosquito 11 fish), feed on mosquito larvae and are used in biological control programs (Sota and Mogi, 12 1992). Additionally, genetically modified mosquitoes are being developed as a novel approach to 13 suppress mosquito populations (Alphey et al., 2002). 14 Environmental management strategies focus on reducing mosquito breeding sites and 15 modifying habitats to make them less suitable for mosquito development. Source reduction 16 techniques involve eliminating or treating standing water where mosquitoes breed, while habitat 17 modification aims to alter environmental conditions to discourage mosquito proliferation 18 (Heintze et al., 2007). 19 Personal barrier measures involve using barriers like window screens, mosquito nets, 20 repellents, or protective clothing to prevent mosquito bites. 21 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 8 Integrated Mosquito Management (IMM) emphasizes the coordinated use of multiple 1 control methods tailored to local conditions. Surveillance, larval control, adult control, and 2 community engagement are key components of IMM programs aimed at reducing mosquito 3 populations and disease transmission (World Health Organization, 2019). 4 2.3 Mosquito Attraction and Repellents 5 Mosquitoes are attracted to their hosts by a combination of visual and chemical cues. 6 Visual cues, such as color, play a role in mosquito attraction. Research suggests that mosquitoes 7 are more attracted to dark colors, possibly due to their increased visibility against contrasting 8 backgrounds (Foster and Hancock, 1994). 9 Chemical cues, including carbon dioxide (CO2) and human body odors, also influence 10 mosquito host-seeking behavior. Mosquitoes are highly sensitive to CO2, which is emitted by 11 humans during respiration and serves as a potent attractant for female mosquitoes seeking blood 12 meals (Takken and Knols, 1999). Additionally, human skin odors, such as lactic acid and 13 ammonia, can attract mosquitoes from a distance (Logan et al., 2008). 14 Various repellents are available to protect against mosquito bites. Synthetic chemicals, 15 such as DEET (N,N-diethyl-meta-toluamide), are widely used and highly effective at repelling 16 mosquitoes (Frances and Cooper, 2005). Plant-derived repellents, such as citronella and 17 eucalyptus oil, offer alternative options for personal protection against mosquito bites (Maia and 18 Moore, 2011). 19 2.4 Mosquito Species and Disease Transmission 20 Different mosquito species exhibit varying degrees of vector competence for transmitting 21 diseases to humans. Aedes aegypti and Aedes albopictus are primary vectors of Dengue fever, 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 9 Zika virus, West Nile virus and Chikungunya virus, with Aedes aegypti being particularly 1 efficient in urban environments (Lambrechts et al., 2010). Anopheles mosquitoes are the main 2 vectors of Malaria, transmitting Plasmodium parasites to humans through their blood meals 3 (Sinka et al., 2012). 4 The relationship between mosquito species and disease transmission is influenced by 5 factors such as vector biology, host preferences, and pathogen development. Understanding the 6 ecology and behavior of mosquito vectors is essential for implementing targeted control 7 measures to reduce disease transmission (Weiss et al., 2019). 8 2.5 Mosquito's oviposition ethology 9 Mosquito oviposition behavior, influenced by various factors including color preferences 10 and environmental cues, plays a crucial role in the transmission of diseases. Different mosquito 11 species exhibit distinct preferences in color for oviposition sites, often correlating with the 12 diseases they transmit. Understanding these ethological preferences is essential for effective 13 vector control strategies and disease prevention efforts. 14 2.5.1 Aedes aegypti: 15 Aedes aegypti is a primary vector of several devastating diseases, including dengue fever, 16 Zika virus, West Nile virus, and chikungunya. Research has shown that Aedes aegypti females 17 exhibit a preference for dark colors such as black and brown for oviposition sites (Bentley and 18 Day, 1989). These dark-colored sites often contain stagnant water, such as discarded containers, 19 which serve as breeding grounds for Aedes mosquitoes and contribute to the transmission of 20 diseases like dengue fever and Zika virus. 21 2.5.2 Aedes albopictus: 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 10 Another important vector species, Aedes albopictus, is associated with the transmission 1 of diseases such as dengue fever, Zika virus, West Nile virus, and chikungunya. Unlike Aedes 2 aegypti, Aedes albopictus displays a preference for lighter colors like white or light blue for 3 oviposition (Koenraadt et al., 2003). These preferences may vary depending on environmental 4 conditions and habitat characteristics, influencing the distribution of oviposition sites and disease 5 transmission patterns. 6 2.5.3 Culex pipiens: 7 Culex pipiens is a widespread mosquito species known for transmitting West Nile virus, a 8 potentially severe neurological disease. Studies have indicated that Culex pipiens females prefer 9 oviposition sites with high organic content, often found in dark-colored water bodies (Rey et al., 10 2006). These preferences contribute to the proliferation of Culex mosquitoes and the 11 transmission of West Nile virus to humans and other vertebrate hosts. 12 2.5.4 Anopheles spp.: 13 Anopheles mosquitoes are vectors of malaria parasites, a life-threatening disease that 14 affects millions of people worldwide. Different species within the Anopheles genus may exhibit 15 varying oviposition preferences. For example, Anopheles gambiae, a major malaria vector in 16 sub-Saharan Africa, shows a preference for dark-colored oviposition sites (Munga et al., 2006). 17 These sites, often found in natural water bodies like swamps and puddles, provide suitable 18 breeding habitats for Anopheles mosquitoes and contribute to the transmission of malaria. 19 In addition to color preferences, environmental factors such as temperature, humidity, and 20 vegetation also influence mosquito oviposition behavior and disease transmission dynamics. 21 Mosquitoes prefer shaded areas with moderate temperatures and high humidity for oviposition, 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 11 as these conditions promote larval survival and development (Lounibos, 2002). Vegetation 1 provides additional shelter and resources, creating ideal habitats for mosquito breeding and 2 disease transmission (Clements, 1999). 3 2.5.5 Oviposition radius 4 Gravid mosquito females exhibit a remarkable ability to search for suitable oviposition 5 sites, often traveling significant distances and enduring varying environmental conditions to lay 6 their eggs in standing water. The duration and distance traveled by gravid females largely depend 7 on factors such as species, habitat availability, and physiological state. For instance, Aedes 8 mosquitoes, vectors of diseases like dengue and Zika, have been observed to travel up to several 9 hundred meters in search of suitable breeding sites, with some individuals capable of flying 10 distances of over a kilometer (Service, 1997). Similarly, Anopheles mosquitoes, responsible for 11 transmitting malaria, are known to actively seek out water bodies for oviposition, often traveling 12 several kilometers from their blood-feeding sites (Clements, 1999). Gravid females may spend 13 several days or even weeks in search of suitable oviposition sites, utilizing various sensory cues 14 and environmental cues to guide their behavior (Clements, 1999). This remarkable behavior 15 underscores the importance of effective mosquito control strategies to target breeding sites and 16 minimize the risk of disease transmission. 17 2.5.6 Mosquito eggs viability 18 Mosquito eggs possess remarkable resilience, capable of surviving in a desiccated state 19 for extended periods. Research indicates that mosquito eggs can remain viable for varying 20 durations without water, depending on species and environmental conditions. For instance, 21 Aedes mosquitoes, known vectors of diseases such as dengue and Zika, have been reported to 22 maintain viability for up to several months under favorable conditions (Christophers, 1960). 23 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 12 Similarly, studies on Anopheles mosquitoes, responsible for transmitting malaria, suggest that 1 their eggs can endure desiccation for several weeks, retaining the potential for hatching upon 2 rehydration (Service, 1997). This adaptive trait enables mosquito eggs to persist through adverse 3 environmental conditions, contributing to the resilience and persistence of mosquito populations. 4 Mosquito eggs are typically laid on the surface of standing water, although some species 5 may deposit them on moist substrates near water bodies. The surface location of mosquito eggs 6 serves several crucial functions in the reproductive process. Firstly, positioning the eggs on the 7 water surface provides protection from drowning and predation, as it allows for easy access to 8 atmospheric oxygen for respiration (Clements, 1999). Additionally, the surface tension of the 9 water supports the buoyancy of the eggs, preventing them from sinking and facilitating their 10 development (Clements, 2000). 11 The exchange of oxygen is vital for the survival and development of mosquito eggs. Like 12 all insect eggs, mosquito eggs require oxygen for respiration during embryonic development. 13 The thin outer layer of the egg, called the chorion, is permeable to gases, allowing oxygen to 14 diffuse into the egg while carbon dioxide is released (Clements, 1999). This exchange of gases 15 through the eggshell is essential for maintaining aerobic respiration and ensuring the viability of 16 the developing embryo. 17 Various household substances have been identified for their efficacy in destroying 18 mosquito eggs, ranging from natural and environmentally friendly options to chemical 19 compounds with larvicidal properties. Soap disrupts water surface tension, suffocating mosquito 20 eggs, while vinegar creates an inhospitable acidic environment hindering egg development. 21 Essential oils like citronella possess larvicidal properties, deterring egg-laying and destroying 22 existing eggs, and diluted bleach solutions disrupt larvae and egg integrity. Additionally, a thin 23 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 13 layer of vegetable oil forms a barrier on water surfaces, suffocating eggs. However, effectiveness 1 can vary based on factors like concentration and application method, emphasizing the need for 2 caution, especially with chemical-based products, to ensure safety for humans and non-target 3 organisms. (Rajapaksa et al., 2014; Clements, 1999; Maia and Moore, 2011) 4 3. MATERIALS AND METHODS: 5 3.1 The Human-Controlled Breeding Site (HBCS) method 6 The HCBS method represents a proactive approach to controlling mosquito populations 7 through targeted intervention at key stages of the mosquito life cycle. This section delineates the 8 step-by-step methodology for implementing the HCBS method, emphasizing its efficacy in 9 attracting gravid female mosquitoes and disrupting the mosquito reproductive process. 10 3.1.1 Deployment of HCBS Devices 11 The first step in the HCBS method involves the strategic placement of HCBS devices in 12 areas susceptible to mosquito breeding in urban and suburban environments. HCBS devices are 13 positioned at floor level to maximize visibility to flying female mosquitoes while remaining 14 inconspicuous to human observation. The devices are distributed evenly across the target area to 15 ensure comprehensive coverage and efficacy in mosquito population control. 16 To minimize breeding site competition effectively, two strategies are essential: (a) the 17 elimination of existing breeding sites, and/or (b) the incorporation of other standing water 18 sources into the HCBS system. In the former approach, the nearest breeding site would be the 19 HCBS device, compelling gravid mosquitoes to utilize it exclusively. Conversely, in the latter 20 approach, regulating potential standing water sources to align with the HCBS methodology 21 transforms them into functional HCBS devices. 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 14 3.1.2 Attracting Gravid Female Mosquitoes 1 Once installed, HCBS devices are designed to attract gravid female mosquitoes seeking 2 oviposition sites for eggs that must be laid. The design features of the HCBS devices, including a 3 shallow basin and a portion of the water surface covered from light, mimic natural breeding 4 habitats favored by mosquitoes. This strategic design encourages female mosquitoes to lay their 5 eggs in the HCBS devices, thereby initiating the mosquito life cycle within a controlled 6 environment. 7 3.1.3 Removal and Treatment of Water 8 After a predetermined period, typically 10 days to coincide with the development of 9 mosquito larvae into pupae, the water from the HCBS devices is carefully removed and 10 transferred to a separate container. This step is crucial since if the water is not removed within 11 this timeframe, mosquito pupae will emerge as adults, rendering the method ineffective and 12 inadvertently contributing to mosquito reproduction. Moving forward, it is imperative to explore 13 the feasibility of automating the water replacement process in HCBS devices to mitigate this 14 issue and ensure the continued efficacy of mosquito population control efforts. 15 3.1.4 Destroying and killing the collected eggs, larvae and pupas. 16 One option to destroy and kill the eggs, larvae and pupas from the HCBS devices, it’s to 17 treat it with household substances known for their larvicidal properties. Common substances 18 such as soap, vinegar, essential oils, or bleach are added to the water to destroy mosquito eggs, 19 larvae, and pupae present in the container. This targeted treatment ensures the eradication of 20 mosquito offspring and prevents their proliferation in the environment (see Section 2.5.5). 21 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 15 It is crucial to emphasize that only water should be poured into the HCBS container. If 1 the mosquito female detects traces of other substances, including insecticides or household 2 products discussed previously, there is a risk that she may reject the HCBS device as an 3 oviposition site. This would render the method ineffective and unsuitable for mosquito 4 population control. 5 In dire scenarios where household substances are unavailable, simply pouring the water 6 from the HCBS device onto a dry surface can effectively eliminate larvae and pupae but not 7 necessary eggs. However, the easiest alternative to kill the individuals on the three stages is just 8 to bury the collected water beneath a few centimeters of soil (in a garden or even a pot), thus 9 ensuring continued mosquito population control. 10 3.1.5 Reinstallation of HCBS Devices 11 Following treatment, the HCBS devices are refilled with fresh water and reinstated in 12 their original location. 13 3.2 Considerations taken during the design of the HCBS Device 14 The design of the Human-Controlled Breeding Site (HCBS) device was informed by a 15 comprehensive literature review on mosquito ethology and its interactions with humans (Section 16 2). Understanding the behavior of mosquitoes and their preferences for oviposition sites needs 17 was crucial in developing an effective breeding site for controlling mosquito populations. 18 3.2.1 Shape Design 19 The HCBS device was designed with a shallow basin to mimic natural breeding habitats 20 favored by mosquitoes. Its shape allows for a relatively large-exposed water surface, providing 21 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 16 ample space for egg deposition. Additionally, the device incorporates a portion of the water 1 surface covered from light, offering protection to mosquito offspring against potential predators. 2 3.2.2 Color Selection 3 Five colors were selected for testing the HCBS device: White, Black, Red, Green, and 4 Blue. Color selection played a significant role in determining the attractiveness of the device to 5 gravid female mosquitoes. The color of the HCBS device was chosen to ensure visibility to 6 mosquito females while remaining semi-hidden to humans. This balance is crucial to ensure that 7 mosquitoes perceive the oviposition site as hidden from predators. 8 3.2.3 Placement and Visibility 9 The HCBS device is ideally located at floor level to ensure maximum visibility from 10 flying female mosquitoes seeking oviposition sites. It must be strategically positioned to be 11 visible to mosquito females while remaining semi-hidden from human view, creating the 12 perception of a secure oviposition place away from predators. 13 3.2.4 Additional Features 14 To facilitate transportation and maintenance, the HCBS device is equipped with a cap or 15 slicing cover to close the exposed water surface when not in use or during harvesting. This 16 feature ensures the integrity of the breeding site and prevents contamination. Importantly, no 17 insecticides or mosquito-killing devices are added to the water within the HCBS to avoid 18 deterring gravid female mosquitoes from ovipositing. 19 3.3 Experiment design 20 The experiment was conducted in Peru, specifically in the department of Lima, within the 21 district of La Molina, which is situated at an altitude of approximately 237 meters above sea 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 17 level. Its geographical coordinates are approximately 12.0760° S latitude and 76.9502° W 1 longitude. This region experiences climatic conditions typical of a coastal desert, characterized 2 by moderate temperatures, high relative humidity, and minimal rainfall. During the months of 3 February to May, which coincide with the austral summer, La Molina typically records average 4 temperatures ranging from 22°C to 27°C. Relative humidity levels vary between 60% to 70%. 5 Rainfall is scarce, with an average total annual precipitation of less than 10 millimeters. 6 The mosquito species commonly found in this area include Aedes aegypti, the primary 7 vector for diseases such as dengue, Zika, and chikungunya. Additionally, Culex quinquefasciatus, 8 known for transmitting West Nile virus, and Anopheles species responsible for malaria 9 transmission, are also prevalent in the region. 10 The experiment was designed to evaluate the efficacy of Human-Controlled Breeding 11 Site (HCBS) devices in attracting gravid female mosquitoes for oviposition, with a focus on 12 color preference. The HCBS devices were specially designed based on findings from the 13 literature review on mosquito ethology and breeding site preferences. Using 3D printing 14 technology, the devices were fabricated in ABS material in five different colors: white, black, 15 red, green, and blue. 16 3.3.1 Installation and Setup 17 All HCBS devices were filled to the middle with potable water and installed in the same 18 location, following the specified placement guidelines to ensure maximum visibility to flying 19 female mosquitoes. After 10 days of exposure to mosquito access, the HCBS devices were 20 capped and removed from their location. To minimize potential biases due to the very specific 21 location of each HCBS device within the installation location, the distribution of colors among 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 18 HCBS devices was randomly altered at each reinstallation. This process was replicated five times 1 to ensure robustness and reliability of the findings. 2 3.3.2 Data Collection and Analysis 3 In the laboratory, a count of larvae and pupae from each HCBS device was conducted to 4 evaluate mosquito attraction. Due to equipment constraints, egg counts were not feasible for this 5 experiment, and therefore, only larvae and pupae were included in the tally. After counting, the 6 HCBS devices were refilled with water and reinstalled in their original location. 7 It's worth noting that in our study, we prioritized assessing the functionality of HCBS 8 rather than eradicating the mosquito population in our test area. Consequently, the eggs, larvae, 9 and pupae were not eradicated after collection; instead, they were transferred to another 10 container to mature into adults and contribute to the ongoing mosquito population in the study 11 area. This approach ensured a consistent supply of adult mosquitoes for testing the effectiveness 12 of HCBS methodology and devices. 13 3.3.3 Laboratory Environment 14 The installation site was situated inside a laboratory exposed to mosquito access, 15 providing a semi-controlled environment for the experiment (conditions similar to those of a 16 room in a house). This setting allowed for consistent conditions across replicates and minimized 17 external variables that could influence mosquito behavior and oviposition decisions. 18 4. RESULTS AND DISCUSSION: 19 4.1 The design 20 Figure 1 shows the final design of the HCBS device used in our study whereas Figure 2 21 shows the different color devices installed during the experiment. 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 19 1 2 Figure 1: Design drawings of the HCBS device, showing (a) top view of the body, (b) side view 3 of the body, (c) front view of the body, and (d) top view of the slicing cap. Measurements are in 4 millimeters (mm). 5 The slicing cap (d) is placed on the top view of the body (a), enabling slicing between 6 both arms for easy opening and closing of the HCBS device. 7 8 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 20 1 Figure 2: Photograph depicting HCBS devices in all colors used during the experiment, 2 installed in their respective locations. 3 4.2 Experiment results 4 4.2.1 Results on the HCBS method 5 The successful deposition of eggs by gravid mosquitoes into the HCBS devices, followed 6 by the destruction of these individuals within the 10-day timeframe, serves as compelling 7 evidence of the efficacy of the system. This crucial demonstration underscores the functionality 8 of both the HCBS method and device. Without the presence of HCBS devices, these eggs would 9 have inevitably hatched, contributing to the burgeoning mosquito population. Thus, the ability to 10 attract gravid mosquitoes and disrupt their reproductive cycle within the designated timeframe 11 serves as a robust validation of the HCBS system's effectiveness. This innovative approach 12 challenges the commonly current recommendation to destroy all possible locations where 13 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 21 mosquitoes can lay their eggs, providing a controlled environment for oviposition within 1 households while simultaneously reducing mosquito populations at the source. 2 4.2.2 Results on the HCBS color device 3 The sampled adult mosquito population from the laboratory revealed that all captured 4 mosquitoes belonged to the Aedes aegypti species. 5 Table 1: Number of mosquito’s larvae and pupas in the HCBS devices by color and date: 6 HCBS device color Harvesting date April 20th April 30th May 10th May 20th May 30th Black 0 0 0 0 0 Blue 0 0 0 0 0 Green 2 8 17 15 12 Red 0 0 0 0 0 White 0 0 0 0 0 7 After tallying the larvae and pupae across all HCBS devices, a significant statistical 8 difference emerged in the number of specimens collected, particularly between the green HCBS 9 device and others of varying colors. This statistical difference arises from the exclusive 10 utilization of the green device by mosquitoes for oviposition, as outlined in Table 1. 11 Contrastingly, the remaining colors did not serve as oviposition sites, leading to a lack of 12 statistical difference among them. Furthermore, the temporal variation in the number of 13 individuals within HCBS devices was influenced by declining temperatures observed during the 14 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 22 final assessment period, impacting not only our laboratory's mosquito population but also that of 1 the broader region. 2 The Human-Controlled Breeding Sites (HCBS) method demonstrates remarkable efficacy 3 in controlling mosquito populations, exerting influence over three of the four life stages of 4 mosquitoes. Our findings confirm that when HCBS is correctly applied, it achieves a 100% 5 effectiveness in disrupting the mosquito life cycle by collecting laid egg and eliminating all 6 together with larvae and pupae previously hatched. 7 One of the key advantages of HCBS is its low implementation cost, which promotes 8 widespread citizen participation in mosquito reduction campaigns. This cost-effectiveness not 9 only encourages community engagement but also facilitates the scalability of mosquito control 10 efforts in urban and suburban areas. 11 In the event of massive HCBS deployment in urban and suburban environments, a 12 significant reduction in mosquito populations can be anticipated. By disrupting reproduction, 13 HCBS ensures that the current adult mosquito population will be the last local generation. Within 14 a short timeframe, typically within 1 week to 10 days, the absence of male mosquitoes prevents 15 fertilization, effectively limiting the population's ability to replenish since only unfertilized 16 female mosquitoes would theoretically remain alive after this period. 17 While the primary focus of HCBS implementation is in urban and suburban areas, its 18 potential applicability in rural settings warrants consideration. Gravid female mosquitoes tend to 19 seek the closest and safest place to lay their eggs. With HCBS devices installed within or around 20 houses, mosquitoes are likely to prefer ovipositing in the HCBS rather than expending energy to 21 find distant natural environments where predators exist (see Section 2.5.5). Although its 22 effectiveness may be somewhat diminished compared to urban areas, HCBS still serves to reduce 23 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 23 local mosquito populations and disrupt the feedback loop of mosquito repopulation in natural 1 environments. 2 Our study also identifies the importance of device color selection in optimizing HCBS 3 effectiveness. Among the colors tested, green HCBS devices consistently demonstrated the 4 highest efficacy in attracting gravid female mosquitoes for oviposition. Additionally, HCBS 5 devices must have a portion of the water surface shielded from light. This feature makes female 6 mosquitoes more likely to select this device for oviposition, as it offers protection for their 7 offspring against predators. This issue was verified during the retrieval and counting of the 8 individuals collected in the laboratory. When the slicing cap of the HCBS device was opened 9 under the lights, all larvae swiftly moved towards the shaded water within the device. This 10 finding underscores the importance of considering mosquito behavior and preferences in the 11 design of effective mosquito control strategies. 12 Overall, the findings of this study underscore the potential of HCBS as a valuable 13 addition to Integrated Mosquito Management strategies, particularly in urban and suburban 14 settings. By targeting multiple life stages of mosquitoes and incorporating features that enhance 15 attractiveness and efficacy, HCBS offers a promising approach to reducing mosquito populations 16 and mitigating the transmission of mosquito-borne diseases in diverse environments. Further 17 research and field trials are warranted to explore the long-term effectiveness and scalability of 18 HCBS implementation in different geographic contexts. Additionally, optimizing the design of 19 HCBS devices warrants attention to maximize their efficacy and applicability. Finally, the HCBS 20

can undergo evaluation for potential application in combating other insect-borne vector 21 diseases. 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 24 One suggestion is to leverage disaster risk management systems capable of widespread 1 communication via SMS text messages to convey crucial information to residents in a specific 2 area. The proposal involves utilizing a country's system to coordinate the simultaneous 3 installation of HCBS devices across the entire community or city. Similarly, on the 10th day, 4 residents would receive alerts prompting them to harvest and reinstall the HCBS devices. This 5 approach ensures uniform implementation of control programs throughout the community, 6 reminding residents simultaneously to remove the devices to mitigate the negative effects 7 discussed in Section 3.3.3. 8 5. CONCLUSIONS 9 The Human-Controlled Breeding Site (HCBS) methodology is a groundbreaking 10 approach that diverges from the common recommendation to eliminate all potential mosquito 11 egg-laying sites. Instead, it creates controlled breeding sites to attract female mosquitoes for 12 oviposition. Since humans oversee these sites, the eggs, larvae, and pupae can be effectively 13 being mechanically destroyed before maturing into adult mosquitoes. HCBS proves to be a 14 highly effective method for controlling mosquito populations due to its ability to intervene in 15 multiple stages of the mosquito life cycle. By targeting three out of the four stages of mosquito 16 development, HCBS offers a comprehensive approach to mosquito control that can significantly 17 reduce mosquito populations and mitigate disease transmission in urban and suburban areas. 18 HCBS provides a controlled environment within households where mosquito females can 19 oviposit their eggs. By attracting gravid females to lay their eggs in the breeding site, HCBS 20 devises effectively intercepts the first stage of mosquito development, preventing eggs from 21 being laid in natural breeding habitats. Considering that the mosquito’s life cycle in a water 22 environment from egg to pupa has a timespan of around 10 days, at the time the HCBS is 23 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 25 harvested, it will contain individuals in all the three stages from eggs to pupas. At the time the 1 water is discharged from the HCBS device, all the individuals collected are killed at once. 2 Although HCBS primarily targets the egg, larval, and pupal stages of the mosquito life 3 cycle, it complements existing mosquito control methods that focus on adult mosquitoes. By 4 intervening in multiple stages of mosquito development, HCBS provides a holistic approach to 5 mosquito control that enhances the effectiveness of Integrated Mosquito Management (IMM) 6 strategies. Additionally, the localized nature of HCBS allows for targeted control efforts in areas 7 with high mosquito abundance or disease transmission, contributing to a more efficient and 8 sustainable mosquitoes control programs. 9 The HCBS device developed in this study played a pivotal role in facilitating the 10 scientific experimentation of the HCBS method, offering insights into critical factors such as 11 color selection for enhancing system efficiency. However, the paramount aspect lies in the 12 adaptability of the HCBS methodology, as any household receptacle capable of retaining water 13 for over 10 days can serve as an HCBS device (e.g., cups, big-mouth bottles, dishes, etc.). When 14 considering alternative household HCBS devices, key considerations include color, shading, and 15 total water surface area exposed to attract gravid mosquitoes for oviposition. It is important to 16 remember that eggs must be laid, and gravid mosquitoes require standing water for this purpose, 17 with no preferred options being available for their reproduction, any source will do. If we control 18 the source, then we control the reproduction of those mosquitoes. 19 In dire scenarios where household substances are unavailable, simply pouring the water 20 from the HCBS device onto a dry surface can effectively eliminate larvae and pupae but not 21 necessary eggs. However, the easiest alternative to kill the individuals on the three stages is just 22 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint 26 to bury the collected water beneath a few centimeters of soil in a garden or pot, ensuring 1 continued mosquito population control. 2 In conclusion, this study presents a promising methodology for mosquito population 3 control through HCBS methodology especially for urban and suburban areas. By providing a 4 sustainable and effective means of reducing mosquito populations, especially in areas endemic to 5 diseases like dengue fever, yellow fever, West Nile virus, Zika, and others, we envision a 6 substantial contribution to global health initiatives. Implementing this innovative approach on a 7 larger scale holds the potential to significantly mitigate the burden of mosquito-borne diseases 8 worldwide, enhancing public health outcomes and promoting sustainable development. 9 COMPETING INTERESTS 10 The authors declare no competing interest. 11 ACKNOWLEDGMENTS 12 The authors acknowledge the use of generative AI technology ChatGPT 3.5. to improve 13 the paper’s reading. 14

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